Hematopoietic stem cells (HSCs) can respond to microenvironmental cues that modulate cell proliferation, differentiation, survival, self-renewal, and migration. These responses are critical for the HSC to contribute to hematopoiesis long-term during steady-state and during stress. Defects in these responses can lead to hematologic disease. The most durable and complete cure for many diseases would be stem cell replacement, however stem cell transplant is currently not warranted for many benign disorders. Understanding the basic biology of HSCs may lead to novel approaches for stem cell transplant and for promoting endogenous hematopoietic recovery following myeloablation. These goals may have significant clinical benefit. Furthermore, the same mechanisms of HSC regulation may be involved in leukemic stem cell biology. Our research during the first cycle of this R01 was focused on defining the role of JAK/STAT signaling in normal HSC biology. This work uncovered roles for STAT5 activation in growth, long-term competitive repopulating activity, and self-renewal of HSCs. We showed that deficiencies in HSCs were almost identical to c-Mpl knockout mice but deficiencies in multipotent progenitor differentiation were considerably greater, illustrating a broad requirement for STAT5 in hematopoiesis. We were surprised to find that a Balb/c derived modifier locus that included the Gab2 gene modulated the steady- state engraftment ability as measured in W/Wv hosts. We now provide preliminary evidence that Gab2 is also required for normal hematopoiesis and it cooperates with STAT5 through a direct interaction. Furthermore, we show that Gab2 and STAT5 cooperate in normal HSC engraftment and that myeloproliferative disease resulting from constitutive activation of STAT5 is attenuated by Gab2 deficiency. We propose the following specific aims to further explore the role of STAT5 activation in hematopoiesis: 1) Explore novel non-myeloablative conditioning based on STAT5/PI3-kinase inhibition using STAT5/Gab2 compound mutant mice. 2) Establish the structure-function relationship for STAT5 in normal hematopoiesis using the complete null STAT5 knockout mouse background as a template. Our preliminary data has established feasibility for this approach using fetal liver retroviral transduction to add back STAT5 mutants lacking specific domain functions. 3) Determine whether survival signaling is the Achilles'heel of constitutive STAT5 activation in leukemic hematopoiesis. We will define mechanisms by which STAT5 acquires survival signals such as Bcl-2 to promote leukemogenesis. This work is expected to significantly advance our understanding of the pivotal role of STAT5 signaling in normal and leukemic hematopoiesis and potentially uncover new therapeutic strategies with fewer side effects based on suppression of specific STAT5 domain functions. It is now unequivocal that the JAK/STAT signaling pathway is defective in many blood diseases including immunodeficiency, autoimmunity, and cancer. We believe that a better understanding of the structure-function mechanisms of STAT5 signaling may have broad application for development of novel therapeutics with fewer side effects.